Impact of Allogrooming in Domestic Cats (Felis silvestris catus) on Mitochondrial DNA Profiling of Shed Hairs

Abstract

Allogrooming is a normal behavior for socially bonded cats. Cat hairs may have epithelial cells on the shaft from at least two cat contributors, the host (groomee) and the donor (groomer). To determine the likelihood of obtaining a mixture or incorrect DNA profile in cat hairs, feline mtDNA control region from hairs of allogrooming cats was isolated and analyzed by direct sequencing. Two DNA extraction methods were tested; hair washes and complete digestion of hairs. For five allogrooming pairs with different mitotypes, thirteen of the 126 sequences (10.3%) matched the mitotype of the groomer, not the groomee. Forty-three sequences (34.13%) suggested the presence of both mitotypes, groomer and groomee. Approximately 2.4% of mtDNA sequences appeared heteroplasmic at mitotype defining sites. Heteroplasmy was not observed in 157 control sequences. Mitotypes from the groomer was 11-fold more difficult to obtain from hairs that were completely digested before DNA isolation and was not observed in samples if the hairs were washed prior to digestion. Unlike contamination issues in human forensic cases, obtaining more than one mtDNA profile from a feline hair sample could narrow the pool of suspects since the implicated cat(s) would have to be within the same vicinity and have social contact.

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12 The Open Forensic Science Journal, 2013, 6, 12-19
1874-4028/13 2013 Bentham Open
Open Access
Impact of Allogrooming in Domestic Cats (Felis silvestris catus) on
Mitochondrial DNA Profiling of Shed Hairs
Robert A. Grahn
*
, Tina I-T Huang and Leslie A. Lyons
Department of Population Health & Reproduction, School of Veterinary Medicine, University of California, Davis,
Davis, CA 95616, USA
Abstract: Allogrooming is a normal behavior for socially bonded cats. Cat hairs may have epithelial cells on the shaft
from at least two cat contributors, the host (groomee) and the donor (groomer). To determine the likelihood of obtaining a
mixture or incorrect DNA profile in cat hairs, feline mtDNA control region from hairs of allogrooming cats was isolated
and analyzed by direct sequencing. Two DNA extraction methods were tested; hair washes and complete digestion of
hairs. For five allogrooming pairs with different mitotypes, thirteen of the 126 sequences (10.3%) matched the mitotype of
the groomer, not the groomee. Forty-three sequences (34.13%) suggested the presence of both mitotypes, groomer and
groomee. Approximately 2.4% of mtDNA sequences appeared heteroplasmic at mitotype defining sites. Heteroplasmy
was not observed in 157 control sequences. Mitotypes from the groomer was 11-fold more difficult to obtain from hairs
that were completely digested before DNA isolation and was not observed in samples if the hairs were washed prior to
digestion. Unlike contamination issues in human forensic cases, obtaining more than one mtDNA profile from a feline
hair sample could narrow the pool of suspects since the implicated cat(s) would have to be within the same vicinity and
have social contact.
Keywords: Forensic science, control region, heteroplasmy, feline, mitochondrial DNA, mitotype.
INTRODUCTION
According to the 2011-2012 National Pet Owners Survey
[1], 38.9 million households own a cat accounting for 86.4
million cats in the United States. Cat hairs are readily
transferred from pet to person, object to person and even
person to person, during everyday activities, such as
grooming, petting, or simply coming into contact with
clothing or furniture. Locard’s exchange principle states that
when two items come into contact with each other, there is
an exchange of materials [2] thus when an individual enters
the home of a pet owner, the likelihood of leaving the house
with hairs from that pet is very high. D'Andrea et al. (1998)
demonstrated that during criminal activity, a perpetrator is
unlikely to leave the house of a pet owner without
inadvertently taking some pet hair [3].
Cat fur is composed of different hair types. Generally,
cats have a longer thick, top guard hair layer and two layers
of shorter and thinner, more abundant undercoat of awn and
down fur [4]. The majority of a cat’s coat and the majority of
shed hairs are from the undercoat. Although cats do have
more pronounced seasonal shedding in the spring and fall,
cats shed hairs throughout the year with each individual hair
having its own growth cycle [5]. For forensic purposes,
microscopic analysis is typically limited to the guard hairs
that contain species-specific diagnostic features. However,
*Address correspondence to this author at the 980 Old Davis Rd, Veterinary
Genetics Laboratory, School of Veterinary Medicine, University of
California – Davis, Davis, CA 95616, USA; Tel: 530-752-2314;
Fax: 530-752-3556; E-mail: ragrahn@ucdavis.edu
all cat hairs are a potential source for DNA and therefore a
means of individual identification.
Cats spend 8% of their waking hours licking their fur to
maintain coat quality by removing ectoparasites [6], dirt, and
stale oil [7]. Mother cats frequently groom their young to
keep them clean. Additionally, cats that are related, mate
with each other or cohabitate will often groom each other
[8]. The common name for cat to cat licking is social
grooming, or allogrooming. There is no differentiation
between sexes for allogrooming with both males and females
equally likely to groom either sex. During grooming, buccal
cells in the saliva may be transferred to the hair shaft. Thus,
cat hair found at a crime scene may actually have epithelial
cells on the hair shaft from two contributors; the host cat, the
groomee, and the donor cat, the groomer.
Mitochondrial DNA (mtDNA) can be more reliably
amplified from hairs than genomic DNA due to limited,
degraded genomic DNA fragments found in hair shaft.
However, successful amplification of small amplicons has
been demonstrated [9, 10]. Reference mtDNA genome
sequences are available for a variety of species [11-20]
including the domestic cat [21]. The cat mtDNA control
region (CR) has been shown to be effective in forensic
diagnostics and a DNA sequence database is available for
comparison [22-25]. Using direct sequencing to mimic field
sample analysis, the frequency of cross-cat DNA transfer to
hairs due to allogrooming was investigated under optimal
circumstances. The sensitivity of the laboratory methods
used for detection of allogrooming was also evaluated.

Impact of Allogrooming in Domestic Cats (Felis silvestris catus) The Open Forensic Science Journal, 2013, Volume 6 13
MATERIALS AND METHODOLOGY
Animals
Thirteen socially bonded domestic cats (Felis silvestris
catus) were evaluated for detectable DNA transfer during
allogrooming behavior. Ten cats were from the University of
California - Davis Feline Genetics Research Colony and
three random bred cats were privately owned. Over the
course of one month, allogrooming hair samples were
collected from the 13 cats, some of which were used in more
than one pairing, yielding nine allogrooming pairings (Table
1).
Table 1. Cat Pairings for Allogrooming mtDNA Analysis
Cat
Lab ID
Groomer or Groomee
Mitotype Pairing
1 4445 Groomer B I
2 8638 Groomer B II
3 5338
Groomer
Groomer
B
III
VIII*
4 8637 Groomer B IV
5 11286
Groomer
Groomee
C
V*
VI*
6 11288
Groomer
Groomee
B
VI*
VII*
7 9969 Groomee B IX*
8 9890
Groomee
Groomer
G
VIII*
IX*
9 8639 Groomee B I
10 5072 Groomee B II
11 10699 Groomee B III
12 9712 Groomee B IV
13 11287
Groomee
Groomer
A
V*
VII*
*The mtDNA mitotypes are different in five cat pairings (V – IX).
Reference DNA Sample Collection & Extraction
Buccal reference samples were collected from each cat
with sterile cytology brushes (Fisher Healthcare, Houston,
TX). DNA was isolated using the QIAamp
®
DNA Mini Kit -
buccal swab protocol (Qiagen, Valencia, CA). Total DNA
concentrations from buccal swab isolations ranged from 2
ng/μl – 19 ng/μl as determined using a NanoDrop ND-1000
Spectrophotometer (NanoDrop Technologies, Inc.,
Wilmington, DE). To confirm the transfer of sufficient DNA
for isolation from a licked surface, cats were presented wet
cat food (BABYCAT INSTINCTIVE, Royal Canin Inc., St.
Charles, MO) on plastic spoons. After the food was
consumed, the licked spoons were air dried and placed in
labeled envelopes at room temperature. Within eight days,
the surface of the spoons was wiped with phosphate buffered
saline moistened Q-tips (Johnson & Johnson, Langhorne,
PA) to liberate transferred buccal cells. DNA was isolated
from the Q-tips using QIAamp
®
DNA Mini Kit - buccal
swab protocol (Qiagen, Valencia, CA).
To determine the detection limit of mixed mtDNA
mitotypes by direct sequencing, reference DNA samples
from three cats with mitotypes A, B and C were quantified
by qPCR using the Applied Biosytems 7300 Real Time PCR
system (Applied Biosystems, Foster City, CA) and dilution
mixtures were constructed in the following ratios: 1:1, 1:2.5,
1:5, 1:10, 1:25, 1:50, 1:100, 1:250, 1:500, and 1:1000. A
total of five ng of template DNA was used in each PCR
reaction at the listed major and minor component ratios.
Reciprocal dilutions were performed to determine if
directional dilution bias existed. Each dilution series was
tested with four replicates.
Allogrooming Hair Sample DNA Extraction
When social allogrooming of co-housed cats was
observed, hair was immediately cut with clean scissors close
to, but not including, the root at the grooming site. Clipped
hairs, which included all three hair types, were then placed in
individual paper envelopes and sealed with tape. Total DNA
was isolated from 20 non-washed individual hairs from each
sample using the QIAamp
®
DNA Mini Kit - buccal swab
protocol (Qiagen, Valencia, CA). This technique does not
fully digest the hair. Extracted total DNA was stored at 4°C.
Hair Digestion Protocol
Twenty hairs from three cat pairings (Table 1, pairs V-VII)
were fully digested for DNA isolation. For each pairing, 10
hairs were washed to remove potential DNA from the external
surface and 10 hairs were unwashed. For the washed hairs,
each hair was inverted for 2 hrs in TE buffer to liberate any
external cells and transferred to a clean tube for digestion.
Each washed and unwashed hair was placed in an individual
1.5ml Eppendorf tube containing 200 l of digestion buffer
(0.039 M DTT, 0.1 M NaCl, 50 g/ml Proteinase K, 2% SDS,
and 0.003 M CaCl
2
in TE (10mM Tris-HCl, pH 8.0, 1.0 mM
EDTA) and incubated with inversion at 56°C overnight. DNA
was isolated by phenol:chloroform extraction using N-butanol
for DNA precipitation [26]. Samples were purified and
concentrated using Microcon Centrifugal Filter Devices YM-
100 (Millipore, Jaffrey, NH) following manufacturer’s
protocol with three additional distilled water rinses to ensure
sample purity. Samples were eluted with 10 – 50 l TE and
stored at -20°C.
PCR Amplification
The mtDNA CR was amplified from the spoon derived
DNA samples, the buccal swab DNA samples and the
digested hair samples as previously described [25]. PCR
amplification was performed on a PTC-200 DNA Engine (MJ
Research, Waltham, MA). Each 20 l reaction mix contained
3 l of template DNA (6-60ng), 1.25 mM dNTPs (Gen Script
Corp., Piscataway, NJ), 1 M each of the forward and reverse
primers (Operon Biotechnologies, Inc., Huntsville, AL), 2 mM
MgCl
2
,
1X PCR Buffer and 0.5 units Taq DNA polymerase
(Denville Scientific Inc., Metuchen, NJ). PCR cycling
conditions were as follows; 94°C for 3 min for denaturation,
35 cycles of 94°C for 1 min, 56°C for 30 sec, and 72°C for 45
sec, followed by a final extension at 72°C for 10 min. Samples
were stored at 4°C until further analysis.

14 The Open Forensic Science Journal, 2013, Volume 6 Grahn et al.
mtDNA Sequencing and Analysis
Mitochondrial DNA control region products were
sequenced in both directions with published primers [25]
using BigDye Terminator v3.1 cycle sequencing kit (Applied
Biosystems, Foster city, CA) according to manufacturer’s
specifications on a 3730 DNA Analyzer (Applied
Biosystems). These primers generate an amplicon avoiding
NUMT background observed in some CR regions of the
feline mt genome [21, 27]. Sequences were compared and
evaluated using Sequencher 4.0™ software (Gene Codes
Corporation, Ann Arbor, MI). All sequences were trimmed
and aligned using both Sequencher and Bioedit (Ibis
Biosciences, Carlsbad, CA) programs to the 402 bp
“Sylvester” reference sequence of the cat mtDNA CR [22,
25]. Sequence sites with more than one nucleotide were
identified and the sequences compared to a cat mtDNA
database to determine the corresponding mitotype [22, 25].
The sequences generated from the hairs were compared to
the reference sequence of each cat. Considering that the hair
samples could be a mixture of mitotypes, consensus contigs
were not constructed from the reverse and forward
sequences, but compared independently to the reference
sequence from the corresponding cat. Excluding areas where
the sequence was aberrant, any sequence position with more
than one nucleotide in the electropherogram was noted,
regardless of amplitude.
RESULTS
Baseline mtDNA Mitotypes
Reference mtDNA sequences of 13 cats were generated
from buccal swab DNA. Ten cats had the same sequence,
mitotype B, for the 402 bp region. Three cats had mitotypes,
A, C, or G (Table 1). The diagnostic sites between these
mitotypes are presented in Table 2. Mitotype pair A – B has
six variant sites, A – C has nine variant sites, B- C has three
variant sites and B – G has one variant site. Comparison of
the mtDNA sequences of the 13 cats revealed that four of the
nine allogrooming pairings had an identical sequence; thus,
the detection of allogrooming could be observed in only five
of the nine pairs (Table 1). The four pairings with identical
mitotypes were used as “controls” for contamination and
heteroplasmy evaluations (presented below). Mitochondrial
DNA was successfully extracted and amplified from the
licked spoons. The spoon DNA mitotypes were identical to
the reference mitotypes derived from the buccal swabs (data
not shown).
Minimal Contribution Detection
PCR amplicons were successfully generated and
sequenced from a minimum of 5 pg/μl of every dilution
mixture. To determine the minimal DNA contribution
necessary to detect allogrooming using direct sequencing
technology in the presence of a primary DNA source,
reciprocal serial dilutions were established for the DNA from
the cat pairings with mitotypes A-B. If an electropherogram
peak was visible above baseline, regardless of relative
amplitude, it was identified as a detected mixture (for
example see Fig. 1C position 16895). Electropherogram
scales were not adjusted to visualize minor peaks. All
amplification and sequencing reactions were repeated to
verify results. Mixtures were detected at all variant sites at
the 1:5 dilution. Diagnostic differences between mtDNA
sequences were consistently detected at the 1:50 dilution,
Table 2. Detection of Minor Component Mitotype by Direct Sequencing
Mitotype Position
U20753 16824A 16986T 63T 130T 159T 173G
Dilution
SRS 11A 173T 260T 327T 356T 370G
A + + + + + +
1:1
B + + + + + +
A + + + + + +
1:2.5
B + + + + + +
A + + + + + +
1:5
B + + + + + +
A + + - + + -
1:10
B + + + + + -
A + + - + + -
1:25
B + + - + + -
A - + - + + -
1:50
B - - - + + -
A - - - + - -
1:100
B - - - - - -
A - - - - - -
1:250
B - - - - - -

Impact of Allogrooming in Domestic Cats (Felis silvestris catus) The Open Forensic Science Journal, 2013, Volume 6 15
although not all possible sites were always observed. The
minor component of a 1:100 dilution was detected in one
sample, and only at one mitotype defining position. The
sensitivity of sequencing data to reveal the minor component
mitotype is presented in Table 2.
Allogrooming Detection
Forty sequences were generated from non-washed hairs
for the nine pairings (N = 360), however 47 sequences
(13.06%) were excluded due to poor amplification. An
additional 30 sequences were excluded in pairings XIII and
IX due to poor sequence quality at the diagnostic sites. The
remaining 283 sequences could be from the groomee, the
groomer, or a mixture of both mitotypes. Four pairings (I -
IV) were between cats with the same mtDNA mitotype.
There were no variable sites detected in these 157 sequences
suggesting no heteroplasmy in this region in the control data
set. Of the five pairings with different mitotypes, 13 of the
126 sequences (10.32%) matched the mitotype of the
groomer cat and the groomee mitotype was not observed
(Table 3). Both mitotypes from a pairing were present in 43
(34.13%) sequences. The remaining sequences (~56%)
matched the groomee mitotypes only (Table 3). A majority
of samples with detectable mixtures showed significant
differences in peak amplitudes (Fig. 1). As observed in the
minimal mixture detection study, although mixtures at
diagnostic mitotype sites were observed in 43 sequences, not
every possible diagnostic site was detected (Fig. 1B vs 1C),
particularly in the 17 samples representing pairing V - VII.
In three mixture sequences (2.4%), peak amplitudes of the
two possible nucleotides at the variant sites were
approximately equal, suggesting heteroplasmy rather than
sample mixture. This observation was limited to pairings
VIII and IX both having only one diagnostic site.
Parenthetical numbers in Table 2 indicate observations of
two nucleotides in the electropherogram in a contiguous
sequence. For example, in pairing V (mitotypes A and C),
the three sequences at position 16985G and the seven in
16986T are all from the seven sequences listed as mixtures.
The 36 representing the groomee showed no mixed sites.
The chance of detection of allogrooming contamination from
immediately isolated, unwashed cat hairs that have not been
fully digested is 44%.
Hair Digestion Analysis
DNA was obtained from 10 washed and 10 unwashed
completely digested hairs for three pairings of cats with
different mtDNA mitotypes (pairings V, VI and VII). For the
Table 3. Nucleotide Identity from Allogrooming Hair Samples
Pairing Source Number Type Position
U20753 16824A 16859T 16985G 16986T 59T 63T 130T 159T 173G
SRS 11A 46C 172A 173T 255C 260T 327T 356T 370G
EU864495.1 A A C A C C T C T A
EU864496.1 B G C A T C A T C G
EU864497.1 C G T G T T A T C G
EU864501.1 G A C A T C A T C G
I-IV Groomee/r 157 B G C A T C A T C G
V Groomee 36 A A C A C C T C T A
V Groomer 0 C G T G T T A T C G
V Mixture 7 A/C *G/A (3) C A/G (3) C/T (7) C/T (6) T/A (7) C/T (6) T/C (7)
#
A/G (4)
VI Groomee 36 C G T G T T A T C G
VI Groomer 4 B G C A T C A T C G
VI Mixture 0 C/B G T/C (0) G/A (0) T T/C (0) A T C G
VII Groomee 20 B G C A T C A T C G
VII Groomer 5 A A C A C C T C T A
VII Mixture 10 B/A G/A (4) C A T/C (10) C A/T (6) T/C (6) C/T (0) G/A (0)
VIII Groomee 7 A G C A T C A T C G
VIII Groomer 0 G A C A T C A T C G
VIII Mixture 12 A/G G/A(12) C A T C A T C G
IX Groomee 5 G A C A T C A T C G
IX Groomer 3 A G C A T C A T C G
IX Mixture 11 G/A G/A(11) C A T C A T C G
*Four of seven mixture sequences failed to produce quality data at this position.
#Three of seven mixture sequences failed to produce quality data at this position.
Parenthesis () indicate number of sequences scored with both nucleotides.

16 The Open Forensic Science Journal, 2013, Volume 6 Grahn et al.
30 hairs washed prior to digestion, 51 of the 60 sequencing
reactions generated passed quality control, including 16, 19,
and 16 sequences from the respective pairings. All generated
sequences matched the groomee only, showing no evidence
of contamination. Fifty-four of the 60 sequences were
successful for the unwashed hairs, including 15, 20, and 19
sequences from the respective pairings. Only pairing V
contained two of 15 sequences that indicated a mixture of
mitotypes (13.33%). All other unwashed hairs had the
groomee mitotype. For completely digested washed and
unwashed hairs, the chance of detecting a mitotype mixture
of allogrooming cats was 0.0% and 3.7%, respectively.
DISCUSSION
Cat hair has had limited use as crime scene evidence,
likely due to the lack of feline forensic expertise, limited
feline population databases and the lack of knowledge of
feline forensic resources. While the morphological traits of
hair can provide insight into the nature of species and/or
breed of the source animal [23], DNA isolated from the hair
can help identify a specific individual by potentially
providing STR, mtDNA and SNP profiles [28-31]. Just as
Locard’s Exchange Principle predicts transfer of evidence
between sources, allogrooming can result in material transfer
between cats, resulting in hairs covered with multiple DNA
sources and potentially a mixture of DNA profiles.
DNA mixture profiles can suggest sample contamination,
potentially leading to the dismissal of genetic forensic
evidence. However, in the case of cat hair, multiple DNA
sources could benefit an investigation as the “contamination
by a second DNA source” may not be due to poor quality
control or improper evidence handling. Multiple sources of
cat DNA on cat hair can likely be a result of allogrooming
[32]. DNA transfer can occur between cats during
courtships, but mating is less frequent than allogrooming and
a cat will likely remove any contaminating DNA from
courtship during incessant self-grooming throughout the day
[7]. For allogrooming DNA transfer to occur, cats must be
familiar with one another, cohabitate, and/or live within the
Fig. (1). Electropherogram data showing amplitude profiles at mitotype defining positions for two extractions of the same sample. A, B and
C show contiguous electropherogram data with homologous stretches removed. Nucleotide positions relative to U20753 are provided at the
top. A) 1:1 mix of a mitotypes A and C mixture sample. Variant site nucleotide amplitudes are approximately the same. B and C) A-groomee
and C-groomer hair sample direct sequence data with differing peak profiles.

Impact of Allogrooming in Domestic Cats (Felis silvestris catus) The Open Forensic Science Journal, 2013, Volume 6 17
same limited territory. Approximately 30% of cat-owning
households own more than one cat. Thus, the presence of
two DNA sources on a cat’s hair can actually narrow a
suspect pool since two cats would need to be implicated and
they would have required the opportunity to allogroom. This
study examined the likelihood of detecting allogrooming
under an ideal ascertainment scenario – immediately
isolating the groomed hairs after witnessing allogrooming
behavior.
To determine the limits of detection of the groomer
mtDNA profile, serial dilutions of groomee vs groomer DNA
were evaluated. Using fluorescence-based direct sequencing
techniques and standard PCR amplification protocols,
allogrooming contamination was consistently detected when
the groomee DNA was mixed with groomer DNA, up to a
dilution of 1:50. Cat hair with DNA contribution from a
groomer less than 1:50 requires more robust sequencing
techniques to identify the contamination. Thus, natural
variation in the amount of DNA source material transferred
during allogrooming may significantly impact secondary
source DNA detection.
When allogrooming occurs between cats, the cats’ own
grooming behavior will likely re-clean the allogroomed areas
when anatomically possible. In this study, allogrooming was
directly witnessed and groomed hairs were immediately cut,
maximizing the potential for the detection of allogrooming
contamination. Hair roots were not included in the analyses,
mimicking shed hairs that are more likely to be found at a
crime scene. Additionally, the inclusion of the root bulb
would likely have overwhelmed the mixture with groomee
DNA, obscuring the groomers DNA signature. In the
allogrooming analysis, hairs were not digested and root
bulbs were not included, thus, the analyzed DNA should
have been derived from external epithelial cells on the hairs.
Cat allogrooming pairings included cats with and without
the same mtDNA haplotype. Although a mixture study
would potentially necessitate cloning of the DNA fragments
to confirm the presence of two mitotypes, this study only
used direct sequencing methods for several reasons. Firstly,
cloning is not routine in casework, and the current study was
designed to mimic forensic laboratory analyses in the
detection of mixtures. Secondly, the mtDNA region analyzed
does not contain a repeat sequence, thus, variation can be
clearly identified by the presence of two nucleotides in a
sequence. Thirdly, the level of detection and level of
contamination from allogrooming was unknown, thus,
extensive sequencing of clones may have been required to
identify a second DNA source contribution especially
considering successful amplification and sequencing of 5pg
in the absence of a competing major template
The control pairings were used to estimate heteroplasmy
and laboratory contamination. Twenty hairs were examined
from each of four pairings of cats that had the same mtDNA
haplotype. At least one forward and reverse sequence was
generated for each replicate, resulting in 157 sequences.
Considering the evaluations of only the major DNA source
from the groomee, these four pairings did not show
heteroplasmy or other sources of contamination based on a
robust sampling of 20 individual hairs per cat. This data
supports that the mtDNA CR region under analysis has low
heteroplasmy (none observed in over 31,500 control base
pairs sequenced in this study), even in tissues with a high
mitotic index [33], such as hair.
Five pairings of cats included individuals with different
mtDNA haplotypes. More than one mitotype was observed
in ~44% of sequences from hair samples removed from
cross-groomed sites, identified by either mixture sequences
or sequences of the groomer. Two peaks were only observed
in electropherograms at mitotype defining positions. In three
of the five pairings, ~10% of the generated sequences
represented the mtDNA mitotype of the groomer, while the
groomee mitotype was not observed. These data suggest that
the DNA extracted from the surface of a cat hair using the
Qiagen extraction protocol can provide additional DNA
typing information and has a high probability of detecting
allogrooming if present but may also provide a cautionary
warning regarding suspect searches derived from hair surface
data.
Most standard operating procedures for hair analysis
include washing the hair, followed by complete digestion
prior to DNA extraction and analysis. An additional aspect
of this study examined washed vs unwashed hairs that were
both digested to completion, all of which lacked hair bulbs.
Of the 51 sequences generated from the 30 washed, digested
hairs, none indicated that two different mtDNA profiles were
present. Of 55 sequences from unwashed hairs, four
sequences (7.2%) – both forward and reverse sequences from
two separate hairs – indicated a mixture. Thus, analysis of
the wash itself may detect mixtures due to allogrooming
more successfully than analysis of unwashed digested hairs
and digestion of washed hairs yields mitotypes of the
groomee only. However, sufficient replicates must be
performed on the wash to confirm the identity and detection
of mixtures, which may prove problematic with limited
samples.
The digested hair evaluation demonstrates that mixtures
can be detected even when groomee DNA is far more
abundant than any transferred DNA. However, an
electropherogram with two nucleotides at a site could be
misinterpreted as heteroplasmy. The relative nucleotide
amplitudes in electropherograms may suggest contamination
vs heteroplasmy. Although the ratio of any two mitotypes in
a heteroplasmic individual is unknown, one might predict
that both templates would be well represented resulting in
base pair data points with similar amplitudes. Allogrooming
DNA transfer would therefore appear as nucleotide peaks
with lower amplitudes on the electropherogram. Only four
sequences (3.2%) suggested heteroplasmy based on
relatively equal peak amplitude while the remainder, ~31%,
appeared to be mixtures with low-level contamination. A
single variable site would be more likely to represent
heteroplasmy than multiple sites in one sequence. However,
the likelihood of heteroplasmy being detected at a mitotype
defining sites is expected to be no more likely than any of
the other 401 positions under evaluation in this study.
Between all currently known common cat mitotypes, nine
sites are variable in the 402 bp region. Hence, the
recognition of a "heteroplasmic" site being at a site of
variation between mitotypes is important to consider when
reporting heteroplasmy vs mixed DNA sources. In
disproportionate mixtures, not all diagnostic sites were
observed, possibly resulting from sequencing chemistry

18 The Open Forensic Science Journal, 2013, Volume 6 Grahn et al.
incorporation bias of adjacent nucleotides. Of true
heteroplasmic sequences, the probability of just one
heteroplasmic site occurring in the human mtDNA CR is
roughly 10% [34-36]. One study suggested six heteroplasmic
sites in a human mtDNA sequence, however these variations
turned out to be sequencing errors [37, 38]. Thus, multiple
site variation more likely represents sequencing errors or
evidence of mixture. The location of observed variation
should be considered especially when occurring at sites of
known variants.
Four CR mtDNA mitotypes are common for cats in the
USA and approximately 10% of cats are unique at the
mtDNA CR [22]. Approximately 7.3% of a groomer and
groomee combination would both have most common
mitotype A, which is found in ~27% of the USA population
(0.27 x 0.27 = 0.073). Considering the cat mitotype
frequencies from California, seven mitotypes account for
~82% of the population, 25 mitotypes account for the
remaining 18% of the population. Considering the seven
pairings (A - A, B – B, C - C) that would have the same
mitotypes, cats would have a probability of 17% to have the
same mitotype, 83% having different mitotypes.
CONCLUSIONS
Hair mtDNA typing can be impacted by the grooming
behaviors of cats. A surface licked by a cat provides
adequate DNA for mtDNA analyses and the food off a spoon
method introduced here can help crime scene investigators
obtain DNA samples from fractious cats. Approximately
34% of the unwashed hair samples obtained from cat
pairings that were known to have allogroomed showed the
presence of more than one DNA source. The evaluation,
through mtDNA analysis, of the wash liquid from a cat hair
sample can add additional information to the mtDNA profile
obtained and should be used to the advantage of criminal
investigators. DNA extraction methods can impact the typing
results that are obtained from hair samples. Unwashed hairs
have the potential to carry more than one DNA source due to
allogrooming, but following washing, digestion of the hair
sample will provide the host mtDNA profile of the
individual cat. Confusion between heteroplasmy and
contamination, either in the laboratory or resulting from
allogrooming, can be clarified by considering the number
and positions of the nucleotide sites of mixtures in
comparison to known cat mitotypes. In most forensics cases,
the amount of evidence that is collected may not provide
more than one or two replicates and suspected contamination
may result in the exclusion of evidence. For the cat, a
secondary DNA source mtDNA profile obtained from a hair
wash may provide valuable additional data to implicate an
appropriate suspect, albeit the cat, the owner, or a situation
supporting hair transfer. Thus, in the cat, multiple DNA
profiles from a DNA source may prove to be of added value
to an investigation and warrants in-depth consideration.
CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
Financial support was provided in part by National
Institutes of Health - National Center for Research Resources
(NCRR) grant R24 RR016094R24, now the Office of
Research Infrastructure Programs (ORIP) grant
R24OD010928, and the UC Davis Forensic Sciences
graduate program. Laboratory assistance, training, and
comments to the manuscript were provided by M.J. Lipinski,
L.H. Bach, J.D. Kurushima, and M. Watters.
REFERENCES
[1] APPA National Pet Owners Survey. Greenwich, CT: American Pet
Products Association, Inc.; 2012, pp. 1-593.
[2] Thornton, J.I.; Kimmel-Lake, D. Trace evidence in crime
reconstruction. In: Crime Reconstruction. 1
st
ed. Chisum W. J.;
Turvey, B. E.; Eds. Elsevier Science & Technology Books,
Philadelphia: 2006, pp. 197-213.
[3] D'Andrea, F.; Fridez, F.; Coquoz, R. Preliminary experiments on
the transfer of animal hair during simulated criminal behavior. J.
For.Sci., 1998, 43(6), 1257-1258.
[4] Searle, A.G.; Jude, A.C. The 'Rex' type of coat in the domestic cat.
J. Genet., 1956, 54(3), 506-512.
[5] WALTHAM. Skin and coat in cats. In: WALTHAM Course on Dog
and Cat Nutrition. Edited by Grandjean D., Butterwick R. (online):
Waltham Centre for Pet Nutrition; 1999, p. 18.
[6] Hart, B.L. Behavioral adaptations to pathogens and parasites - 5
Strategies. Neurosci. Biobehav. Rev., 1990, 14(3), 273-294.
[7] Eckstein, R.A.; Hart, B.L. The organization and control of
grooming in cats. Appl. An. Beh. Sci., 2000, 68(2), 131-140.
[8] The signaling repertoire of the domestic cat and its undomesticated
relatives. In: The domestic cat: the biology of its behaviour. Turner,
D.C.; Bateson, P.; Eds. 2
nd
ed. Cambridge University: Cambridge,
2000, pp. 76-87.
[9] Hellmann, A.; Rohleder, U.; Schmitter, H.; Wittig, M. STR typing
of human telogen hairs--a new approach. Int. J. Legal Med., 2001,
114(4-5), 269-273.
[10] Muller, K.; Brugger, C.; Klein, R.; Miltner, E.; Reuther, F.;
Wiegand, P. STR typing of hairs from domestic cats. For. Sci. Int.
Genet. Sup. Ser., 2008, 1(1), 607-609.
[11] Anderson, S.; Bankier, A.T.; Barrell, B.G.; Debruijn, M.H.L.;
Coulson, A.R.; Drouin, J.; Eperon, I.C.; Nierlich, D.P.; Roe, B.A.;
Sanger, F.; Schreier P. H.; Smith A. J. H.; Staden R.; Young, I.G.
Sequence and organization of the human mitochondrial genome.
Nature, 1981, 290(5806), 457-465.
[12] Kim, K.S.; Lee, S.E.; Jeong, H.W.; Ha, J.H. The complete
nucleotide sequence of the domestic dog (Canis familiaris)
mitochondrial genome. Mol.Phyl.. Evol., 1998, 10(2), 210-220.
[13] Arnason, U.; Gullberg, A.; Janke, A.; Kullberg, M. Mitogenomic
analyses of caniform relationships. Mol. Phylogenet. Evol., 2007,
45(3), 863-874.
[14] Hou, W.R.; Du, Y.J.; Chen, Y.; Wu, X.; Peng, Z.S.; Yang, J.; Zhou,
C.Q. Nucleotide sequence of cDNA encoding the mitochondrial
precursor protein of the ATPase inhibitor from the giant panda
(Ailuropoda melanoleuca). DNA Cell Biol., 2007, 26(11), 799-802.
[15] Lin, C.S.; Sun, Y.L.; Liu, C.Y.; Yang, P.C.; Chang, L.C.; Cheng,
I.C.; Mao, S. J.; Huang, M.C. Complete nucleotide sequence of pig
(Sus scrofa) mitochondrial genome and dating evolutionary
divergence within Artiodactyla. Gene, 1999, 236(1), 107-114.
[16] Park, Y.C. The complete mitochondrial genome sequence of the
Amur leopard cat, Prionailurus bengalensis euptilurus. Mito. DNA,
2011, 22(4), 89-90.
[17] Wei, L.; Wu, X.; Zhu, L.; Jiang, Z. Mitogenomic analysis of the
genus Panthera. Sci. China Life Sci., 2011, 54(10), 917-930.
[18] Willerslev, E.; Gilbert, M.T.; Binladen, J.; Ho, S.Y.; Campos, P.F.;
Ratan, A.; Tomsho, L.P.; da Fonseca, R.R.; Sher, A.; Kuznetsova
T.V.; Nowak-Kemp, M.; Roth, T.L.; Miller, W.; Schuster, S.C.
Analysis of complete mitochondrial genomes from extinct and
extant rhinoceroses reveals lack of phylogenetic resolution. BMC
Evol. Biol., 2009, 9(95), 1-11.
[19] Wu, G.S.; Yao, Y.G.; Qu, K.X.; Ding, Z.L.; Li, H.; Palanichamy,
M.G.; Duan, Z.Y.; Li, N.; Chen, Y.S.; Zhang, Y.P. Population
phylogenomic analysis of mitochondrial DNA in wild boars and
domestic pigs revealed multiple domestication events in East Asia.
Genome Biol., 2007, 8(11), 1-12.

Impact of Allogrooming in Domestic Cats (Felis silvestris catus) The Open Forensic Science Journal, 2013, Volume 6 19
[20] Zhang, W.; Yue, B.; Wang, X.; Zhang, X.; Xie, Z.; Liu, N.; Fu, W.;
Yuan, Y.; Chen, D.; Fu, D.; Zhao, B.; Yin, Y.; Yan, X.; Zhang, R.;
Liu, J.; Li, M.; Tang, Y.; Hou, R.; Zhang, Z. Analysis of variable
sites between two complete South China tiger (Panthera tigris
amoyensis) mitochondrial genomes. Mol. Biol. Rep., 2011, 38(7),
4257-4264.
[21] Lopez, J.V.; Cevario, S.; Obrien, S.J. Complete nucleotide
sequences of the domestic cat (Felis catus) mitochondrial genome
and a transposed mtDNA tandem repeat (Numt) in the nuclear
genome. Genomics, 1996, 33(2), 229-246.
[22] Grahn, R.A.; Kurushima, J.D.; Billings, N.C.; Grahn, J.C.;
Halverson, J.L.; Hammer, E.; Ho, C.K.; Kun, T.J.; Levy, J.K.;
Lipinski, M.J.; Mwenda, J.M.; Ozpinar, H.; Schuster, R.K.;
Shoorijeh, S.J.; Tarditi, C.R.; Waly, N.E.; Wictum, E.J.; Lyons,
L.A. Feline non-repetitive mitochondrial DNA control region
database for forensic evidence. For. Sci. Int. Genet., 2011, 5(1), 33-
42.
[23] Halverson, J.L.; Basten, C. Forensic DNA identification of animal-
derived trace evidence: Tools for linking victims and suspects.
Croat. Med. J., 2005, 46(4), 598-605.
[24] Halverson, J.L.; Lyons, L.A. Forensic DNA Identification of Feline
Hairs: Casework and a Mitochondrial Database. Proc. Am. Acad.
For.Sci., 2004, X, B150.
[25] Tarditi, C.R.; Grahn, R.A.; Evans, J.J.; Kurushima, J.D.; Lyons,
L.A. Mitochondrial DNA sequencing of cat hair: an informative
forensic tool. J. For. Sci., 2011, 56 (Suppl. 1), S36-46.
[26] Higuchi, R.; von Beroldingen, C.H.; Sensabaugh, G.F.; Erlich, H.
A. DNA typing from single hairs. Nature, 1988, 332(6164), 543-
546.
[27] Goios, A.; Carvalho, A.; Amorim, A. Identifying NUMT
contamination in mtDNA analyses. For. Sci. Int., Genet. Sup.Ser.,
2009, 2(1), 278-280.
[28] Graham, E.A.M. DNA reviews: hair. For. Sci. Med. Pathol., 007,
3, 133-137.
[29] Bowling, A.T. Historical development and application of molecular
genetic tests for horse identification and parentage control.
Livestock Prod. Sci., 2001, 72(1-2), 111-116.
[30] Bender, K.; Schneider, P.M. Validation and casework testing of the
BioPlex-11 for STR typing of telogen hair roots. For. Sci. Int.,
2006, 161(1), 52-59.
[31] Muller, K.; Klein, R.; Miltner, E.; Wiegand, P. Improved STR
typing of telogen hair root and hair shaft DNA. Electrophoresis,
2007, 28(16), 2835-2842.
[32] Crowell-Davis, S.L.; Curtis, T.M.; Knowles, R.J. Social
organization in the cat: a modern understanding. J. Feline Med.
Surg., 2004, 6(1), 19-28.
[33] Paneto, G.G.; Martins, J.A.; Longo, L.V.; Pereira, G.A.; Freschi,
A.; Alvarenga, V.L.; Chen, B.; Oliveira, R.N.; Hirata, M.H.;
Cicarelli, R.M. Heteroplasmy in hair: differences among hair and
blood from the same individuals are still a matter of debate.
For.Sci. Int., 2007, 173(2-3), 117-121.
[34] Brandstatter A.; Parson, W. Mitochondrial DNA heteroplasmy or
artefacts - a matter of the amplification strategy? Int.J. Legal Med.,
2003, 117(3), 180-184.
[35] Melton, T.; Dimick, G.; Higgins, B.; Lindstrom, L.; Nelson, K.
Forensic mitochondrial DNA analysis of 691 casework hairs. J.
For. Sci., 2005, 50(1), 73-80.
[36] Melton, T.; Nelson, K. Forensic Mitochondrial DNA Analysis:
Two Years of Commercial Casework Experience in the United
States. Croat.Med. J., 2001, 42(3), 298-303.
[37] Budowle, B.; Allard, M.W.; Wilson, M.R. Critique of interpretation
of high levels of heteroplasmy in the human mitochondrial DNA
hypervariable region I from hair. For.Sci. Int., 2002, 126(1), 30-33.
[38] Grzybowski, T. Extremely high levels of human mitochondrial
DNA heteroplasmy in single hair roots. Electrophoresis, 2000,
21(5), 548-553.
Received: March 12, 2013 Revised: August 27, 2013 Accepted: September 11, 2013
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